The present invention relates to methods for determining an effect of a Clostridial toxin upon a muscle. In particular, the present invention relates to use of a dermal topography method for determining an effect of a Clostridial toxin upon a facial muscle.
Movement of the face can be due to contractions of muscles underlying the skin and different muscles can move different parts of the face. For example, elevation of the brow results from contraction of the frontalis muscle. Electromyographic methods have been used to study the activity of various facial muscles. See e.g. Fridlund A. et al., Guidelines for Human Electromyographic Research, Psychophysiology 1986; 23(5): 567-590; Vitti M, et al., Electromyographic Investigation of Procerus and Frontalis Muscles, Electromyogr. clin. Neurophysiol. 1976,16: 227-236, and; Tassinary L. et al., A Psychometric Study of Surface Electrode Placements for Facial Electromyographic Recording: I. The Brow and Cheek Muscle Regions, Psychophysiology 1989; 26(1): 1-16.
In particular, electromyography, including surface electromyography (sEMG) has been used to investigate activity of the frontalis muscle and resultant brow displacement. See e.g. van Boxtel A, et al., Amplitude and bandwidth of the frontalis surface EMG: Effects of electrode parameters, Psychophysiology 1984; 21(6): 699-707, and; Pennock J. D., et al., Relationship between muscle activity of the frontalis and the associated brow displacement, Plast Reconstr Surg November 1999; 104(6): 1789-1797.
Additionally, it is known to study skin topography by making a silicone rubber negative replica (a mold) of a skin surface area. The mold captures three dimensional details of the skin surface and computerized image analysis of skin line density, depths and length analysis shown can be carried out thereon. Grove, G. L., et al, Objective method for assessing skin surface topography noninvasively, chapter one, pages 1-32 of Cutaneous Investigation in Health and Disease, edited by Leveque J-L., Marcel Dekker, Inc. (1989). This method has been used to study how micro-furrows on the forearm can increase in depth from about 33 xcexcm in children to up to about 100 xcexcm in the elderly. Corcuff P. et al., Skin relief and aging, J Soc Cosmet Chem 1983; 34:177-190. The same silicone rubber impression method has been used to examine the effect of a topical cream to treat photodamaged skin, as by reduction of periorbital (crows feet) wrinkles. Leyden J. J., et al., Treatment of photodamaged facial skin with topical tretinoin, J Am Acad Dermatol 1989; 21(3) (part 2): 638-644, and; Grove G. L., et al., Skin replica analysis of photodamaged skin after therapy with tretinoin emollient cream, J Am Acad Dermatol 1991; 25(2) (part 1): 231-237.
The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals known as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
Botutinum toxin type A is the most lethal natural biological agent known to man. bout 50 picograms of botulinum toxin (purified neurotoxin complex) type A1 is a LD50 in mice. One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18-20 grams each. Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. The botulinum toxins apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine.
Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A has been approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus, hemifacial spasm and cervical dystonia.
1Avaible from Allegran, Inc. of Irvine, Calif. under the tradeneme BOTOX(copyright). 
Botulinum toxin type B has also been approved by the FDA for the treatment of cervical dystonia. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about three months.
Although all the botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes.
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C1 is apparently produced as only a 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin types E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture.
It has been reported that botulinum toxin type A has been used in clinical settings as follows:
(1) about 75-250 units of BOTOX(copyright) per intramuscular injection (multiple muscles) to treat cervical dystonia;
(2) 5-10 units of BOTOX(copyright) per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);
(3) about 30-80 units of BOTOX(copyright) to treat constipation by intrasphincter injection of the puborectalis muscle;
(4) about 1-5 units per muscle of intramuscularly injected BOTOX(copyright) to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid.
(5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 units of BOTOX(copyright), the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired).
(6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX(copyright) into five different upper limb flexor muscles, as follows:
(a) flexor digitorum profundus: 7.5 U to 30 U
(b) flexor digitorum sublimus: 7.5 U to 30 U
(c) flexor carpi ulnaris: 10 U to 40 U
(d) flexor carpi radialis: 15 U to 60 U
(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX(copyright) by intramuscular injection at each treatment session.
It is also known that injection of a botulinum toxin into facial muscles can, by weakening the injected muscles, result in a decrease of hyperkinetic wrinkles in the skin overlying the paralyzed muscles. See e.g. Carruthers A. et al., The treatment of glabellar furrows with botulinum A exotoxin, J Dermatol Surg Oncol 1990 January; 16(1):83.
It is known to use a botulinum toxin to treat: intrathecal pain (see e.g. U.S. Pat. No. 6,113,915); paragangliomas (see e.g. U.S. Pat. No. 6,139,845); otic disorders (see e.g. U.S. Pat. No. 6,265,379); pancreatic disorders (see e.g. U.S. Pat. Nos. 6,143,306 and 6,261,572); migraine (see e.g. U.S. Pat. No. 5,714,468); smooth muscle disorders (see e.g. U.S. Pat. No. 5,437,291); prostate disorders, including prostatic hyperplasia (see e.g. WO 99/03483 and Doggweiler R., et al Botulinum toxin type A causes diffuse and highly selective atrophy of rat prostate, Neurourol Urodyn 1998; 17(4):363); autonomic nerve disorders, including hyperplasic sweat glands (see e.g. U.S. Pat. No. 5,766,606); wound healing (see e.g. WO 00/24419); reduced hair loss (see e.g. WO 00/62746); skin lesions (see e.g. U.S. Pat. No. 5,670,484), and; neurogenic inflammatory disorders (see e.g. U.S. Pat. No. 6,063,768).
Additionally it has been disclosed that targeted botulinum toxins (i.e. with a non-native binding moiety) can be used to treat various conditions (see e.g. U.S. Pat. No. 5,989,545, as well as WO 96/33273; WO 99/17806; WO 98/07864; WO 00/57897; WO 01/21213; WO 00/10598.
A botulinum toxin has been injected into the pectoral muscle to control pectoral spasm. See e.g. Senior M., Botox and the management of pectoral spasm after subpectoral implant insertion, Plastic and Recon Surg, July 2000, 224-225.
Both liquid stable formulations and pure botulinum toxin formulations have been disclosed (see e.g. WO 00/15245 and WO 74703) as well as topical application of a botulinum toxin (see e.g. DE 198 52 981).
Typically, a Clostridial toxin, such as a botulinum toxin, is administered locally and directly into a target tissue, such as a skeletal muscle, by intramuscular or subcutaneous injection. Entry of a Clostridial toxin into the circulatory system is undesirable, since botulism or tetanus can result. Additionally, entry of a Clostridial toxin into the systemic circulation typically results in generation of antibodies against the toxin. The presence of antibodies leads to a loss or diminishment of a desired clinical response, such as a muscle paralysis. Thus, methodologies for determination of bioavailability of a Clostridial toxin practiced in regard to an intravenously or orally administered pharmaceutical are neither relevant nor applicability with regard to a locally (i.e. intravenous or subcutaneous) administered Clostridal toxin.
Unfortunately, therefore methodologies which examine a physiological fluid (i.e. blood, urine) are of little or no value to determine bioavailability of a Clostridial toxin to a target muscle or muscle group, due to the local (non-systemic) administration and effect of the toxin. Thus, currently available analytical techniques to perform classical absorption, distribution, biotransformation and elimination studies on an oral or intravenously administered drugs cannot be used.
Botulinum toxin has been injected into facial muscles, such as the orbicularis oculis, corrugator supercilii and frontalis muscles for the cosmetic purpose of reducing certain facial wrinkles, and it is known to use electromyographic and/or photographic techniques to assess the efficacy of such injections. Guerrissi J. et al., Local injection into mimetic muscles of botulinum toxin A for the treatment of facial lines, Ann Plast Surg 1997; 39(5):447-53. Electromyography has also been used to assess the effect of injection of a botulinum toxin into the sternocleidomastoid muscle for treatment of cervical dystonia. Dressler D. et al., Electromyographic quantification of the paralysing effect of botulinum toxin in the stemocleidomastoid muscle, Eur Neurol 2000; 43:13-16. In sEMG the surface electrodes are placed at fixed distances from the injection point, typically 1 cm and 3 cm from the injection point. The surface electrodes can be used to measure the amplitude and area of a compound muscle action potential (CMAP) during maximal voluntary contraction of the injected muscle. One expects to find that CMAP decreases with the onset of muscle paralytic effect and to increase as the paralytic effect wears off.
Unfortunately, electromyographic methods for determining an effect of a Clostridial toxin, such as a botulinum toxin, upon a muscle or muscle group can be unsatisfactory because of the variability of electrical activity from a particular muscle between patients an even with the same patient in different positions or on different days due to the known vagaries of electrophysiology. For example, repeat surface electromyographic recordings can show significant (i.e. from about 7% to about 20%) variability when taken from the same patient at the same time. Additionally, the extent of maximal voluntary contraction, at which the sEMG recording is taken, can be variable between and among patients.
Photographic methods, such as digital image analysis, have been used to determine efficacy of a botulinum toxin to treat hyperkinetic facial lines. Heckmann M., et al., Quantification of the efficacy of botulinum toxin type A by digital image analysis, J Am Acad Dermatol 2001; 45: 508-514. As with electromyographic methods, photographic methods also show significant intra and inter subject variability. Thus, photographic methods for determining an effect of a Clostridial toxin, such as botulinum toxin, upon a muscle or a muscle group can lack precision and accuracy and the quality and value of the images obtained are as variant as the lighting conditions, type of film used, film speed and the film development process used.
Thus both electromyographic and photographic methods for assessing an effect of a botulinum toxin upon a muscle have significant drawbacks and deficiencies and neither of these methods can readily provide a three dimensional permanent record amenable to analysis.
What is needed therefore is a non-invasive method for determining a pharmacodynamic effect (such as a muscle paralytic effect) of a Clostridial toxin, such as a botulinum toxin, upon a muscle or muscle group, which method provides an accurate and precise three dimensional record amendable to computerized analysis.
My invention fulfills this need and provides a non-invasive method for determining a pharmacodynamic effect (such as a muscle paralytic effect) of a Clostridial toxin, such as a botulinum toxin, upon a muscle or muscle group. Additionally, my method provides an accurate and precise three dimensional record amendable to computerized analysis. The method disclosed herein can comprise the steps of administering a Clostridial toxin to a muscle; making an impression of a feature of a skin surface in proximity to the muscle to which the Clostridial toxin was administered; examining the impression, and; determining onset of paralysis, peak paralysis and duration of paralysis of the muscle by the Clostridial toxin.
The administering step can be carried out by intramuscular injection or subcutaneous injection of the Clostridial toxin. Alternately, a suitable controlled release implant, containing a Clostridial toxin, can be inserted under the skin or within the muscle. Preferably, the muscle is a facial muscle (such as a frontalis muscle) because facial skin can show a more determinable response to injection of a Clostridial toxin into the muscle which underlies the skin. In other words, the skin of the face such as on the forehead has a topography which encompasses easily discernable wrinkles, furrow and lines which can produce a quantifiable response to an intramuscular toxin injection. Thus, a causal connection exists between the paralytic effect of a Clostridial toxin upon a muscle and change in facial topography. I have discovered how to quantify this causality so as to determine pharmocodynamic effects of a Clostridial toxin upon muscles.
Preferably, the Clostridial toxin is a botulinum toxin (such as a botulinum toxin type A, B, C, D, E, F or G) because several botulinum toxins are commercially available and have been used clinically to paralyze various muscles. An embodiment of the present invention encompasses use of from about: 1 unit to about 1,000 units of a botulinum toxin type A (i.e. between about 1-300 units of the BOTOX type A botulinum toxin or between about 1-1000 units of the DYSPORT type botulinum toxin); 10 to 10,000 units of a type B botulinum toxin (such as the MYOBLOC type B botulinum toxin), and; amounts of the other botulinum toxins based on their known differing potencies.
The impression step can comprise applying a polymeric material to the skin surface to thereby obtain a mold which has, on the surface of the mold in contact with the skin surface, a negative replica of a skin surface topography. The examining step can comprise illuminating the negative replica surface of the mold with incident light.
Additionally, the determining step can further comprise determining an extent of diffusion of the Clostridial toxin in the muscle to which the Clostridial toxin was dministered and into a surrounding area. And the determining step can comprise, subsequent to the illuminating step, the step of generating an optical image of the illuminated negative replica surface. Furthermore, the determining step can comprise, subsequent to the generating step, the step of computing a parameter of a skin line present on the negative replica surface.
A detailed embodiment of the present invention is a method for determining a paralytic effect of a botulinum toxin (such as a botulinum toxin type A) upon a facial muscle by: (a) administering a botulinum toxin to a facial muscle by intramuscular injection; (b) making an impression of a feature of a skin surface in proximity to the muscle to which the Clostridial toxin was administered; (c) examining the impression, and; (d) determining onset of paralysis, peak paralysis and duration of paralysis of the muscle by the Clostridial toxin. This method can further comprising the steps of making an electromyographic recording of electrical activity of the facial muscle and photographing the skin surface.
A further detailed embodiment of the present invention is a method for determining a pharmacodynamic effect of a botulinum toxin upon a facial muscle, the method comprising the steps of: (a) administering a botulinum toxin to a facial muscle by intramuscular injection; (b) making an electromyographic recording of electrical activity of the facial muscle; (c) photographing a skin surface in proximity to the muscle to which the Clostridial toxin was administered; (d) making an impression of a feature of the skin surface; (e) examining the impression, and; (f) determining onset of paralysis, peak paralysis and duration of paralysis of the facial muscle by the Clostridial toxin.
The route of administration and amount of Clostridial toxin administered can vary widely according to the particular muscle being injected and various patient variables including size, weight, age, disease severity and responsiveness to therapy. Method for determining the appropriate route of administration and dosage are generally determined on a case by case basis by the attending physician. Such determinations are routine to one of ordinary skill in the art (see for example, Harrison""s Principles of Internal Medicine (1997), edited by Anthony Fauci et al., 14th edition, published by McGraw Hill). Treatment is carried out so as to substantially avoiding entry of the toxin into the systemic circulation (i.e. by use of subcutaneous or intramuscular injection as opposed to intravenous administration).
The specific dosage appropriate for administration is readily determined by one of ordinary skill in the art according to the factors discussed above. The dosage can also depend upon the size of the muscle to be treated or denervated, and the commercial preparation of the toxin. Generally, it is known that the amount of a Clostridial toxin (such as a botulinum toxin) to be injected is proportional to the mass and level of activity of the muscle tissue to be treated.
The present invention includes within its scope the use of any Clostridial toxin which has a long duration therapeutic effect. For example, neurotoxins made by any of the species of the toxin producing Clostridium bacteria, such as Clostridium botulinum, Clostridium butyricum, and Clostridium beratti can be used or adapted for use in the methods of the present invention. Additionally, all of the botulinum serotypes A, B, C, D, E, F and G can be advantageously used in the practice of the present invention, although type A is the most preferred serotype, as explained above.
xe2x80x9cLocal administrationxe2x80x9d means direct injection of the Clostridial into the muscle, subcutaneous or intradermal injection. Systemic routes of administration, such as oral and intravenous routes of administration, are excluded from the scope of the present invention.
The Clostridial toxin (such as a botulinum toxin) used in the present invention botulinum toxin can be a modified Clostridial toxin, that is the toxin can have at least one of its amino acids deleted, modified or replaced, as compared to a native Clostridial toxin. Thus, the Clostridial toxin used can be a recombinant produced Clostidial (i.e. botulinum) toxin or a derivative or fragment thereof.